Preparation of liquids of graded density



United States Patent Oilfice 3,%,i39 Patented Oct. 23, 1962 3,060,130PREPARATION OF LIQUIDS OF GRADED DENSITY Lawrence E. Sacks, Berkeley,Calif., assiguor to the United States of America as represented by theSecretary of Agriculture Filed July 19, 1960, Ser. No. 43,965 3 Claims.(Cl. 252315) (Granted under Title 35, US. Code (1952), see. 266) Anon-exclusive, irrevocable, royalty-free license in the invention hereindescribed, throughout the World for all purposes of the United StatesGovernment, with the power to grant sublicenses for such purposes, ishereby granted to the Government of the United States of America.

This invention relates to and has among its objects the provision ofnovel means and methods for preparing graded density liquids. Furtherobjects and advantages of the invention will be evident from thefollowing description taken in connection with the annexed drawing.

In the drawing:

FIGURE 1 is aside view, partly in cross-section, of an embodiment ofapparatus useful in carrying out the process of the invention.

FIGURE 2 is a side view in cross-section illustrating formation of awedge of solidified liquid of selected density.

In carrying out certain microbiological investigations it is oftendesirable to provide a body of liquid having a graded density, that is,a density which varies from top to bottom of the body of liquid. Suchmaterials are useful, for example, in isolating different microbialspecies from a mixture thereof. To this end, within a centrifuge tube isdeposited a liquid of increasing density, considered in the directionfrom top to bottom. A specimen of the microbial mixture is placed at thetop of the liquid and the tube then centrifuged. After this operation,the tube is examined and it will be seen that microbial species ofditferent intrinsic densities are located at different levels of theliquid. These separate deposits of microbial material can then beisolated from one another by careful decanting, pipetting, or the like.

In the prior art the usual way of establishing the liquid of varyingdensity involves the following steps: A series of solutions areprepared, each having a different density as determined by theconcentration of selected soluble salts therein. The individualsolutions are then poured one after another into the centrifuge tubestarting with the solution of greatest density. This technique hasseveral disadvantages. One is that extreme caution must be observedduring the pouring operation lest the individual solutions be mixed.This would, of course, destroy the density gradient pattern. Anotherpoint is that the finished product has a density pattern which variesstep-wise rather than continuously. For this reason it is difiicult toget clean-cut separations of samples differing by a small degree ofdensity.

In accordance with the present invention the above disadvantages areeliminated. Liquids having continuouslyvarying density gradients mayreadily be prepared and the problem of careful pouring is eliminated.Other advantages of the invention will be apparent from the followingdetailed description of a preferred embodiment of the invention.

In applying the process of the invention, a pair of solutions ofdifferent density is first prepared. The particular densities selectedwill depend on the microbial species or other materials to be separated.Adjustment of density of each solution may be carried out in knownmanner, for example, by adding to water the required proportion of asolute, such as a mineral salt, sucrose, glycerine, etc. The solutionsare warmed and into them is incorporated an amount of a gelling agentsufiicient to cause them to gel when cooled. The t me of gelling agentis not a critical item and any of the materials known to have suchproperties may be used. Typically, one may employ gelatine, agar, algin,gum arabic, or similar heat-reversible gelling agents, that is,materials that form gels that may be re-liquefied by heating.

In the next operation, the two solutions are formed into a compositestructure having two complementary wedge shaped blocks of the solidifiedsolutions. To this end, there is provided a conventional hypodermicsyringe with the needle removed. The stem end of the syringe is cappedand the syringe is about half-filled with the denser solution. Thesyringe is then tipped on its side and held in such position until thesolution is cooled and solidified. FIGURE 2 illustrates this step in theprocess. In this figure syringe 1, provided with cap 2, is shown in thetipped position which is retained while the solution is cooled andsolidified, forming wedge shaped block 3 of solidified solution. Havingprepared the syringe with solidified block '3 in place, the syringe isthen turned upright and an equal amount of the other solution added. Thesyringe is held in upright position until the second solution is cooledand solidified forming wedge shaped block 4 (FIG. 1).

The resulting composite structure of abutting blocks (3, 4) ofsolidified solutions is then subjected to progressive melting to yieldthe solution of graded density. This step may conveniently be carriedout in the device shown in FIG. 1. The illustrated device includes acylinder 5, proportioned to hold syringe 1 snugly. The cylinder isprovided with a handle 6 and an aperture 7, the latter serving to allowprojection of the stem of the syringe. About cylinder 5 is a jacket 8 ofinsulating material in which is embedded heating coil 9. The individualturns of coil 9 are arranged so that they are more concentrated at thelower end of the device, thus to furnish most of the heat output nearthe base of syringe 1. Terminals 10 and 11 are provided for connectionof coil 9 to a suitable source of electrical energy.

In operating the melting device, syringe 1 containing the solidifiedblocks 3, 4 is placed within cylinder 5 such that, when viewed inhorizontal planes, the cross section of block 3 increases and that ofblock 4 decreases progressing from top to bottom, as shown in FIG. 1.The heating coil 9 is then energized. As the system heats up, theportion of solidified material near the base of syringe 1 melts and thesolution runs out of the stem of the syringe. The first increments ofefiiuent thus removed will obviously be of high density because theycontain a high proportion of liquid derived from the denser liquid 3. Insucceeding increments, the density of the effiuent will progressivelydecrease. The effiuent, as it is removed from the syringe, is receivedin a conventional centrifuge tube. During the operation a slightpressure may be maintained on the composite block of solidified materialby applying a finger or a weight on piston 12, thus to keep thesolidified block seated against the base of the syringe and hence ensurethat the melting will take place mainly at the base of the block ofmaterial. It is evident that as the process continues with meltingtaking place mainly at the base of the syringe, the effluent solutionwill gradually change in density. Initially, it will contain a maximumproportion of solution of block 3 with a minimum proportion of solutionof block 4. Later, these proportions will approach equality. Then thesituation will be reversed and solution 4 will become the majorconstituent until the final drop when the effluent contains a maximum ofsolution of block 4 and a minimum of solution of block 3. It is thusapparent that the solution received in the centrifuge tube held belowthe syringe will be in prime condition for isolating particles ofdifferent density as it consists of a liquid, the density of whichvaries continuously from top to bottom of the tube. During theprogressive melting, the amount of heat applied to the system ispreferably controlled so that the efiiuent emerges at a slow rate sothat it may be received in a centrifuge tube with minimum mixing of oneportion of the efliuent with successive portions.

Having prepared a centrifuge tube containing the graded densitysolution, the material to be separated is placed on top of thissolution. The tube is then centrifuged at a temperature high enough thatthe solution remains liquid. After centrifugation the tube may berecooled to solidify the contents. A pinhole is then formed in the baseof the tube and the solid block of solution blown out by air pressure.The block of solution may be cut into sections for isolation of theparticles located in different areas of the solidified block. Thismethod of isolating the different particles constitutes a more selectiveand accurate technique than methods which involve pipetting of differentportions from a mass of the material in liquid form.

It is obvious that it is within the ambit of the invention to carry outthe various steps of the invention in alternate ways. For example, inpreparing the solidified blocks 3, 4, these solidified blocks of liquidmay be cast separately or jointly in molds designed for the purpose,then placed in a syringe or other apertured vessel for progressivemelting. Also, instead of being cast into the desired shape, the Wedgeshaped blocks or blocks of other suitable shapes of varyingcross-section may be cut from large masses of the solidified solutions.

In the preferred modification the blocks of solidified liquid aresubjected to heating directed mainly at the bottom of the compositestructure of the abutting blocks as this structure moves down so thatits base is kept in the region where most of the heat is applied, Inthis Way the melting may be considered as progressing from the bottom ofthe structure upwardly to the top of the structure. However, it isentirely within the purview of the invention to hold stationary thestructure of the complementary wedges while moving a heat-source, suchas an electrically heated coil. progressively upward from bottom to topof the structure, or even in the reverse direction. Moreover, if theabutting blocks are laid on their sides the source of heat may be movedhorizontally from one end of the structure to the other.

Having thus defined the invention, what is claimed is:

1. The method which comprises providing a first solution containing agelling agent, providing a second solution containing a gelling agentand having a density different from the first solution, solidifying andforming the solutions into a composite structure containing a block ofsolidified first solution and an abutting block of solidified secondsolution, the cross section of one of said blocks increasing as it isviewed in horizontal planes progressing from top to bottom of thestructure, the cross-section of the other block decreasing as it isviewed in horizontal planes progressing from top to bottom of thestructure, applying heat mainly to the bottom of said structure to causeprogressive upward melting thereof, and removing the increments ofliquid formed by the melting.

2. The method which comprises providing a first gellable liquid and asecond gellable liquid of density different from that of the firstliquid, gelling and forming the solutions into a composite structure ofa pair of abutting complementary blocks each formed from one of saidliquids, the cross-section of one of said blocks increasing as it isviewed in horizontal planes progressing from top to bottom of thestructure, the cross-section of the other block decreasing as it isviewed in horizontal planes progressing from top to bottom of thestructure, progressively melting the said structure starting from thebottom thereof and continuing upwardly and removing the increments ofliquid formed by the melting.

3. The method which comprises providing a first gellable liquid, formingand gelling said liquid into a first solid block of such dimensions thatthe cross-section thereof increases as it is viewed in horizontal planesprogressing from top to bottom thereof, providing a second gellableliquid of density different from that of the first liquid, forming andgelling said second liquid into a second solid block abutting said firstblock, the cross-section of the second block decreasing as it is viewedin horizontal planes progressing from top to bottom thereof, applyingheat mainly to the bottom of said blocks to cause progressive upwardmelting thereof, and removing the increments of liquid formed by themelting.

References Cited in the file of this patent UNITED STATES PATENTS

1. THE METHOD WHICH COMPRISES PROVIDING A FIRST SOLUTION CONTAINING AGELLING AGENT, PROVIDING A SECOND SOLUTION CONTAINING A GELLING AGENTAND HAVING A DENSITY DIFFERENT FROM THE FIRST SOLUTION, SOLIDIFYING ANDFORMING THE SOLUTIONS INTO A COMPOSITE STRUCTURE CONTAINING A BLOCK OFSOLIDIFIED FIRST SOLUTION AND AN ABUTTING BLOCK OF SOLIDIFIED SECONDSOLUTION, THE CROSS SECTION OF ONE OF SAID BLOCKS INCREASING AS IT ISVIEWED IN HORIZONTAL PLANES PROGRESSING FROM TOP TO BOTTOM OF THESTRUCTURE, THE CROSS-SECTION OF THE OTHER BLOCK DECREASING AS IT ISVIEWED IN HORIZONTAL PLANES PROGRESSING FROM TOP TO BOTTOM OF THESTRUCTURE, APPLYING HEAT MAINLY TO THE BOTTOM OF SAID STRUCTURE TO CAUSEPROGRESSIVE UPWARD MELTING THEREOF, AND REMOVING THE INCREMENTS OFLIQUID FORMED BY THE MELTING.